High precision isotope ratio mass spectrometry (IRMS) of the light elements (C, H, N, 0, S) is an increasingly indispensable tool in many areas of biology, in part due to the advent of the continuous flow approach in the last 15 years. This proposal is a competitive renewal for a program that has integrated novel IRMS instrument development with biomedical applications. The proposed work will capitalize on the interdisciplinary team in place to further extend IRMS into areas of biomedicine where it uniquely applies. Two thrust areas are described, one in analytical development, and the other in isotope physiology. Plans for analytical developments are 1) extension of high precision position-specific (intramolecular) isotope analysis (PSIA) to the isotopes of N; 2) automation of PSIA to permit rapid and standardized sample analysis; 3) continuing development of chemical methods to enhance analyte volatility that are compatible with high precision isotopic analysis, particularly for amino acids. Plans for isotope physiology studies focus on the first attempts to separately evaluate the effects of isotopic inputs and physiological fractionation by holding one constant while investigating the other. Study 1 is a tracer experiment to determine the in vivo turnover of long-lived components of long-lived cells in guinea pigs, particularly purines and pyrimidines, histone amino acids, and fatty acids in neural, liver, and adipose tissue. This experiment also tests the hypothesis that these compounds are a record of isotopic conditions prevailing during development or, in the case of fatty acids, during recent months. Study 2 uses a model microorganism, Paracoccus denitrificans, to investigate the effects of growth conditions and substrates on intrinsic molecular and intramolecular isotope ratios, while holding input isotope ratios constant. Study 3 returns to guinea pigs and investigates the effects of various physiological treatments during pregnancy on molecular and intramolecular isotope ratios in the aforementioned biomolecules in pups. Test physiological states will be induced by the following gross dietary metabolic interventions during pregnancy: basal, high and low protein diets, and mild malnutrition. The successful accomplishment of these plans will yield new tools and experimental approaches for investigation of the impact of developmental factors and recent physiological history on development of chronic disease.